1. Field of the Invention
The present invention relates to variant proteins and polypeptides which manifest enhanced affinity for immobilized-metal affinity matrices. More particularly, the present invention relates to proteins and polypeptides having at least one metal-chelating amino acid sequence engineered thereinto.
2. Prior Art
When recombinant proteins or polypeptides are expressed in bacterial cells and subsequently grown in fermentation tanks, there remains the problem of recovering the desired product in pure form. Immobilized-metal affinity chromatography or ligand exchange chromatography is a well known technique for amino acid and protein purification. F. Helfferich, Nature 189, p. 1001 (1961); H. F. Walton et al, Recent Developments in Separation Science, Vol. VI, Chapter 5 1981. Porath et al, Nature (London), 258, p. 598 (1975); Biochemistry, 22, p. 1625 (1975) and Sulkowski, Trends in Biotechnology, 3, p. 1 (1985), have shown that this technique is well suited for selective fractionation of native proteins according to their content of exposed histidine residues. The chelating ligands such as iminodiacetic acid were bound covalently to oxirane-activated agarose, and the resulting gels were charged with metal ions such as Cu.sup.2+, Zn.sup.2+, Ni.sup.2+ or Co.sup.2+. Such resins have since been utilized for purification of several native peptides and proteins. See, for example, Nilsson et al, Embo J. 4, p. 1075 (1985).
It was desirable, however, that a method be developed for purifying all recombinant proteins and polypeptides, not just those which inherently contain one or more exposed histidine side chains or residues. To this end, hybrid proteins were developed wherein the coding sequence of a protein of interest was fused with the coding sequence of a small histidine-containing peptide. The coding sequence of the affinity peptide is fused to the protein of interest along with the sequence of a specific cleavage site. Such fusion proteins can then be purified by taking advantage of the binding of the histidine-containing peptide, or affinity tail, to the affinity matrix. After purification of the fusion protein, the affinity tail is split off at the designed cleavage site, which is a very difficult and expensive procedure, and the protein of interest is purified in a final step. See, for example, Smith et al Biol. Chem. 263, 7211 (1988). In general this technique is not suited for separating the desired protein from other proteins which inherently include one or more metal-binding amino acids, particularly since many protein impurities are likely to contain multiple exposed histidines. However, if the affinity tail contains 6-8 or more histidines, this method can be useful. See Hochuli et al, Biotechnology, 6, 1321 (1988 ).
Thus, it would be desirable to have a method for protein and polypeptide purification which is applicable to all recombinant proteins and polypeptides and which is effective in separating the desired protein from other proteins which inherently include one or more metal-binding amino acids.
It has now been discovered that the binding of a protein or polypeptide to an immobilized-metal affinity matrix can be enhanced to an extent which is significantly greater than that of native proteins and polypeptides which inherently include one or more metal-binding amino acids as well as fusion proteins and polypeptides. Such enhanced affinity is effected by engineering into such protein or polypeptide a metal-chelating amino acid sequence within the primary sequence thereof and at a specific segment of the secondary structure.